SEPARATION. IDENTIFICATION AND CHARACTERIZATION OF "SOME MYOFIBRILLAR PROTEINS Thesis for the Degree of Ph’. D. MICHIGAN STATE, UNIVERSITY JAMES HENRY RAMPTON 1969 ' imam This is to certify that the thesis entitled SEPARATION, IDENTIFICATION AND CHARACTERIZATION OF SOME MYOFIBRILLAR PROTEINS presented by James Henry Rampton has been accepted towards fulfillment of the requirements for Ph.D. degree in FOOd Science flm (7/4”;wa Major vprofessor Date Méy 9. 1969 0-169 LIBRARY Michigan State University BOOK BRIBERY IIIII (in. 0' BINDING BY IIIIAC 8: SIIIIS' I... DU mun: DC ABSTRACT SEPARATION, IDENTIFICATION AND CHARACTERIZATION OF SOME MYOFIBRILLAR.PROTEINS By James Henry Rampton The purposes of this investigation were to: (l) evaluate different methods for the extraction and purification of myofibrillar proteins, (2) obtain information about the interaction of myofibrillar proteins and their participation in the actomyosin complex, and (3) determine the effect of common meat spoilage bacteria on myofibrillar proteins. The salt soluble proteins from skeletal muscle were separated and characterized.by gel filtration, density gradient centrifugation, ion exchange chromatography and disc gel electrophoresis. Gel filtration and density gradient centrifugation afforded only crude fractionation, while both ion exchange chromatography and disc gel electrophoresis in the presence of 7 M urea separated Weber-Edsall extract into 8-11 principal fractions. Isolation and.purification of known myofibrillar proteins by several methods demonstrated that many so-called "pure preparations" contained significant amounts of contaminants requiring special techni- ques for removal. Myosin, actin and tropomyosin were each prepared by four different methods, and the purest preparations utilized in subsequent studies. The purest preparation.of'myosin was Obtained by chromatography on DEAE- Sephadex ArSO. Disc gel electrophoresis indicated that myosin gave several bands between Rm values of 0.00 and 0.15--the monomers and aggregates apparently remaining at or near the origin and the dissociated polypeptide James Henry Rampton subunits occurring between 0.05 and 0.15. The purest preparation of actin was isolated directly from myofibrils, and gave a single diffuse band at Rm = 0.39 on disc gel electrophoresis. The purest preparation of tropomyosin was Obtained by ammonium.sulfate fractionation and isoelectric precipitation followed.by further purification with DEAE—cellulose chroma- tography in the presence of 0.01 M EDTA. Disc gel electrophoresis of tropomyosin prepared in this way gave two bands at Rm = 0.34 and 0.50, the slow moving component being identified as the oxidized form and the other as reduced tropomyosin. TrOpomyos in was prepared by two methods, while ac-actinin, Q-actinin, inhibitory factor and the extra protein group were each prepared by one method. None of these fractions could be identified by the disc gel system. However, results tentatively indicate that extra.protein Fraction I.A and troponin may be identical.. Weber-Edsall extracts of washed muscle residue or of prepared myofibrils contained myosin, actin, oxidized and reduced tropomyosin, extra protein Fraction I.A, and probablycx-actinin. Specific staining of electrophoretic gels from Weber-Edsall extracts indicated that nucleic acids are complexed with tropomyosin, extra protein Fraction I A, and probably with actin. Actomyosin preparations contained.myosin, actin, reduced trepo- myosin, varying amounts of extra protein Fraction I A, and probably tx-actinin. Gel filtration of actomyosin gave two peaks with apparent molecular weights of 6,000,000 and 50,000,000. The former peak contained mainly actin, myosin, reduced trepomyosin and an unidentified component at Rm = 0.60, while the latter peak consisted mainly of actin and myosin, apparently aggregated together. Perphosphate decreased the sedimentation James Henry Rampton rate of all detectable protein moieties of actomyosin. Sedimentation behaviors indicated that pyrophosphate actually dissociated myosin from the actomyosin complex, yet left actin and tropomyosin in a partially dissociated or otherwise unnatural state. BDTA appeared to have a shmilar action to pyrophosphate on actomyosin, resulting in dissociation of myosin and.partial dissociation of actin and trOpomyosin. Further, EDTA influenced the properties of extra protein Fraction I.A. Micrdbial growth caused no apparent changes in the myofibrillar proteins. However, storage at temperatures above freezing and bacterial growth both decreased the concentration of certain nonrprotein ultra- violet absorbing components detectable by density gradient centrifugation of weber-Edsall extract. SEPARATION, IDENTIFICATION AND CHARACTERIZATION OF SOME MYOFIBRILLAR PROTEINS By James Henry Rampton A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Food Science 1969 ACKNOWLEDGEMENTS The author is deeply indebted to Dr. A..M. Pearson for his encouragement and direction throughout this study and.during prepar- ation of this dissertation. Appreciation is expressed to Dr. R. A. Fennell for his guidance and for the use of his laboratory facilities during specific staining tests. The author also wishes to express appreciation to Dr. J. R. Brunner, Dr. P. K. Kindel and Dr. B. S. Schweigert for their advice and guidance regarding methods of approach and laboratory procedures used during this study. The author is also grateful to Dr. J. F. Price and his laboratory staff for preparing the sterile and inoculated micro-biological samples. Sincere thanks go to my wife, Jackie, whose unceasing support and sacrifice greatly contributed to the completion of this research. Gratitude is also expressed to my parents for their moral and material assistance during this work. ii TABLE OF CONTENDS Page ACKNOWLEDGEMENTS . . ......................... ii LIST OF TABLES . ............... . .......... vi LIST OF FIGURES . ..... . ................... vii (IMFTER I nommuxnm . ...... ... ... ... ... ... .. 1 II REVIEW OF LITERATURE .................... 3 Structure of Skeletal Muscle ...... . ......... 3 Proteins of thelMyofibril. . . ........... 5 The Protein Composition of the Myofibril ..... . . . . . 6 wosm O O O O O O I O O C I O O O O 0 O O O O O O O O O 6 ACtin o o o o o o o I 0000000000000000000 10 Tropomyosin B ...................... l4 Troponin . ...................... . l6 d'aCtinin o o o I o oooooo o cccccccccccc 18 P-actinin ....................... . 19 Inhibitory Factor .................... 20 Extra Protein Group .............. . . . . . 20 Nature of the Actomyosin Complex ............. . 21 Composition . . . . . ., ....... . ....... 21 Effect of ATP and EDTA ................. . 21 Bacterial.Action on Meat Proteins .............. 23 III EXPERIMENTAL METHODS . . ._ .......... . . . . . 25 Sample Preparation ................ . . . . . 25 Rabbit Longissm Dorsi . . ... ..... . ..... . 25 Removal 0 arcop as cFraction ...... . . . . . . 26 WOfibrils I O O O O I O C 010'. O O .......... 26 Salt-Soluble Fraction .............. . . . . 27 Actomyosin . . . ..... . ............. . . 27 Natural Actomyosin ....... . . . . . . . . . . . . 28 iii CHAPTER IV Individual Myofibrillar Proteins ........ . ...... NUosin . . . . . . . .7 ................. Actin . . . . ...................... Tropomyosin B . . . . . ..... . . . . . . . . . . . . Troponin (ESF) ..................... 4- act 1n1n . ....................... 9-actinin . . . ...... . . . . . . . . . . . . . . Inhibitory Factor (IF) . . . . ..... . ....... Extra Protein . . . . . . ..... . . . . . . . . . . . Preliminary Experiments . . . . . . . . . . . . . . . . . . . Salt-Soluble Fraction . . . . . . . . . . . .-. . . . . . Gel Filtration ..................... Sucrose Density Gradient Centrifugation ....... . . Electrophoresis . . . . . . . . . . . . . . . . . . . . . Column Chromatography . . . . . . . . . . . . . . . . . Techniques Utilized in Studying Myofibrillar Proteins . . . . Gel Filtration ..... . .'. . . ...... Sucrose Density Gradient Centrifugation . . . . . . . . . Disc Acrylamide Gel Electrophoresis ........... Absorption Spectra ...... . . . ......... . Nitrogen Determination . . ...... . . . . . . . . . Identification of Fractions . . . ., ........ _. . . Staining . . . . . . . . . . . . ............ Bacterial Action on Myofibrillar Proteins .......... Sample Preparation ..... . . . ..... -. -.- . . . RESULTS AND DISCUSSION ................... Characterization and.Analysis Of Known Proteins ....... Myosin.... ..... ....... Actin ......... ,° . . ... . . . . ..... . . . Tromnlyosjn B. O O O O O O O ,0 O O O O O O I O O O O I O - Troponin (ESF). , OI-Actinin, B -Actinin and Inhibitory Factor (IF). 0 O O O I .. O I O O O O O O O ‘0 O _O O 0 Extra Protein . . . . . . . . .. ...... . Interrelationships of Various Proteins .. . ., ...... Total Extractable Myofibrillar Proteins .......... -. Studies on Actomyosin . . . . . . . -,- . . . . . . . . . . . Composition of Actomyosin . ... . . .p. . . . . . . . . . Composition of Natural Actomyosin (NAM) . . . . ..... Effect of Pyrophosphate on Actomyosin ......... . Effect of EDTA on Actomyosin . . . . . . . .~. . . . . . Bacterial Action oanyofibrillar Proteins . . . ._. . . . . . iv 102 102 111 111 115 117 V SWY o o cccccc o o o o o o o o o o o o o 0 ago 9 o o LIST OF TABLES TABLE Page I Sumnary of Behavior of Protein Preparations . . . . . . . . . 88 II Microbiological Experiments . . . . . . . . . . . . . . . . .119 vi Figure 3C 3D 10 11 12 13 14 15 16 LIST OF FIGURES Gel filtmtion 0f WEDGI'Edsal]. ‘ GXtraCt o o o o o o o .0 o 0 Density gradient centrifugation of weber-Edsall extract . . Gel filtration of 24 ml of weber-Edsall preparation on sephadex G - 1 5 I I I I I I I I I I I I I I I I I I I I I I I Chromatography of breakthrough peak from Figure 3A.on ECTEOLA'CGIIUIOSB o o o o 0 o o I o o o o o o o o o o o o o o Chromatography of breakthrough peak from Figure 3B on AE-ceIIUIOSe I I I I I I I I I I I I I I I I I I I I I I I Chromatography of breakthrough peak from Figure SC on cellulose phOSphate o o o o o o o o o 6 o o o o o o o o o o 0 Purification of myosin on a combination column of DEAE-cellulose and cellulose phosphate (Harris and Suelter, 1967) I I I I I I I ' I I I I I I I I I I I I I I I I I I I I Density gradient centrifugation oflflw . . . . . . . . .>. . . Serial disc gel electrophoretic analyses of the myosin purification shown in Figure 4. . . . . . . . . . ... . . . Disc gel electrophoresis of myosin . . . . . . . . . . . .1 Purification of'myosin on DEAE-Sephadex A-50 (Richards gig-1;... 1967).. o o o 0 up. 0.0 o_o o o o o 0‘. o o o o o o 0 Disc gel electrophoresis of PM, RM fraction 1 and RM fraCtim 2 o o o o o '0 o .0 0 To a o o o o o o o o o o ‘0 0 Purification of actin on Sephadex G-200 (Adelstein et al., 1963) o o o o 0'. o c 0‘. 0‘0 0‘0 0'. 0'. o o o“. oft-7f. 0 Serial disc gel electrophoretic analysis of the actin purification shown in Figure 10 . . .-. ... ... . . . . Density gradient centrifugation of actin. ..... ,. . . . . Disc gel electrophoresis of actin .‘. .-. . . ... ._. . . ._. Density gradient centrifugation of tropomyosin . . .~. . . Disc gel electrophoresis of tropomyosin . . ._. . . . . ... Density gradient centrifugation of tropomyosin . . . . . . . vii Page 41 41 46 46 . -47 47 57 57 S8 58 61 65 65 66 67 67 70 70 Figure Page 17 Disc gel electrophoresis of tropomyosin . . . . . . . . . . . 72 18 Density gradient centrifugation of tropomyosin . . . . . ... 72 19 Disc gel electrophoresis of tropomyosin . . . . . . . ... . . 73 20 Purification of tropomyosin on DEAE- cellulose as adapted from the method ef Davey and Gilbert (1968) . . . _. . . . . . 74 21 Disc gel electrophoresis of tropomyosin.. . . . . ._. . . . . 76 22 Density gradient centrifugation of ESF . . . . . . . . . . . 76 23 Disc gel electrophoresis of ESF . . ... . . . . . . .-. . . . 78 24 Purification of ESF on Sephadex G-200 (Azuma and Watanabe, 1965b) 0 o o 0'. o‘- o o o c o o 0‘. o o a o o o o o o o o o 78 25 Serial disc gel electrophoretic analysis of the purification shown in Figure 24 . . . . .1. . ..... . . . 80 26 Density gradient centrifugation ofcr-actinin. . .-. . . . . . 80 27. .Disc gel electrophoresis of a-actinin . . . . . . . . . . . . 81 28 Density gradient centrifugation of B-actinin. . . . . . . . . 81 29 Disc gel electrophoresis of B-actinin . . . . . . . . . . . . 82 30 Disc gel electrophoresis of IF preparation . . . . . . . . . 82 31 Chromatography of extra protein on DEAE-cellulose (Perry and Zydowo, 1959a) . . . ... . . . . . . . . . . . . . 84 32 Serial disc gel electrophoretic analysis of the separation shown in Figure 31 . . . . . . . . . . . ... .1. . 85 33 Disc gel electrophoresis of sarcoplasmic fraction . . . . . .- 85 34 Ultraviolet absorption spectra of protein preparations . . .- 92 35 Gel filtration of Weber-Edsall extract . . . . . . . . . .'. 94 36 Density gradient centrifugation of weber-Edsall extract . . . 96 37 Disc gel electrophoresis of Weber-Edsall extract . . . . . . 98 38 Disc gel electrophoresis of‘Weber- Edsall extract of I . WOfibrils I I I I I I I I I .I I I I I I I I I II I I I I I I 98 39 Absorption spectrum of extra protein fraction IA . . . . . . 100 40 Disc gel electrophoresis of weber-Edsall extract . . . . . . 103 41 Disc gel electrophoretic comparison of weber- Edsall eXtraCt and aCtGMYOSin o o o o o o o o o o o o o o o o o 0'. 103 42 Gel filtration of actomyosin . . . . . . ._. .~. ... . . . . 105 43 Serial disc gel electrophoretic analyses of the gel filtration pattern from actomyosin shown in Figure 42 . . . 105 viii Figure 44 45 46 47 48A 48B 48C 48D 48E 48F 48G 48H 481 49 Page Density gradient centrifugation of actomyosin . . . . . . .8. 107 Serial disc gel electrophoretic analyses of the density gradient separations of actomyosin shown in Figure 44 . . . 108 Disc gel electrophoretic comparison of actomyosin and mtural aCtOHTYOS in o o o o o o o o '0 o o o o o o o '0 0 go o o 112 Typical gel filtration pattern obtained from weber-Edsall extract of muscle samples in all microbiological experiments 112 Typical density gradient centrifugation pattern obtained fromeeber-Edsall extract of fresh control muscle samples . . 121 Typical density gradient centrifugation pattern Obtained from weber-Edsall extract of control muscle samples after 20 days incubation at either 3 or 10°C . . . . . . . . . . . 121 Typical denisty gradient centrifugation pattern obtained fromeeber-Edsall extract of fresh inoculated muscle samples 122 Density gradient centrifugation pattern obtained from inoculated sample in Experiment No. l (inoculation performed with Streptococcus faecalis) after 20 days incubation at 3°C. . ...... . .8. . . . . . . . . . . . 122 Density gradient centrifugation.pattern obtained from inoculated sample in Experiment No. 2 (inoculation performed with Pediococcus cerevisiae) after 20 days at 3°C. ......... . ..... . . . . . . . . . . . . 123 Density gradient centrifugation pattern obtained from inoculated sample in Experiment No. 3 (inoculation performed with mixed culture from commercial meat) after 20 days of incubation at 3°C . . . . . . . . . . . . . . . . 123 Density gradient centrifugation pattern Obtained from inoculated sample in Experiment No. 4 (inoculation.performed with mixed culture from commercial meat) after 20 days of incubation at 3°C . . . . . . . . . . . . . . . . . . . . . . 124 Density gradient centrifugation pattern Obtained from inoculated sample in.Experhment No. 5 (inoculation perfbrmed with Pediococcus cerevisiae) after 20 days at either 3 or 10°C . . . . . . . . ... . . ... . . . . . . ... . . . . 124 Density gradient centrifugation pattern obtained from inoculated sample in.Experiment No 6 (inoculation performed ‘with Pseudomonas fluorescens) after 20 days of incubation at either 3 or 10°C . . . . . . . . . .~. . . . . . . . . . . 125 Disc gel electrophoresis of Weber-Edsall extracts Obtained during microbiological experiments No. 7 and 8 (Table II). . 127 ix INTRODUCTION Both the meat scientist and muscle chemist are concerned with identifying and characterizing. the proteins of the myofibril. In meat science these proteins may play a key role in determining meat quality, while in muscle chemistry they appear to form the link in the transfer of chemical energy into muscle contraction. Banga and A. Szent-GyOrgyi (1942) distinguished between myosin (myosin A) and actomyosin (myoSin B). This was followed by the discovery and description of actin by Straub (1942) . Since that time myofibrillar proteins have been the subject of intensive study, however, fundamental questions about their identity, properties, and interactions remain unanswered. In recent years, all of the supposedly pure preparations of myo- fibrillar proteins have been found to contain considerable amounts of contaminating substances, as well as lesser amounts of previously unknown factors. Furthermore, the highly complex interactions of the myofibrillar proteins and the different physical properties of impure preparations have renewed interest in the true properties of the pure proteins. Iso- lation of the various pure preparations and studying their interactions should prove beneficial in elucidating their relationships to the physical pmperties of meat and meat products. Since muscle proteins may play a key role in the Spoilage of meat, an understanding of the proteins involved and the nature of the changes could lead to better methods of preventing meat spoilage. The present investigations were undertaken to separate, identify, and purify the myofibrillar proteins, including special studies on the actomyosin complex and its behavior in the presence of pyrophosphate and EDTA. In addition, the nature and extent of degradation of the consti- tuents of the myofibril by the common microbial spoilage flora were also investigated. REVIEW OF LITERATURE Structure of SkeletaL Muscle The structure of skeletal muscle and its relationship to myofi- brillar proteins has been discussed by Bendall (1964) and Lawrie (1966) . They stated that skeletal muscle is surrounded by an outer layer of connective tissue called the epimysium. Branching inward from the epi- mysium are septa of connective tissue, which penetrate the muscle and separate the fibers into bundles. According to Lawrie (1966) these septa are collectively called the perimysium and carry the larger blood vessels and nerves into the muscles. He further stated that the endomysium originates at the perimysium and is composed of septa, which run through- out the muscle bundle to eventually surround each fiber. Lawrie (1966) “stated that the muscle fiber is the essential struc- tural unit of all muscles. He then described the muscle fiber as. a long, narrow, multinucleated cell, which may run from one end of the muscle to the other. He further stated that a fiber may be 10-100p in diameter and up to 34 cm. long. Fibers at the end of the muscle blend in with the endomysium, perimysium, and epimysium, which in turn converge to form tendons. A double membrane called the sarcolemna surrounds each muscle fiber just beneath the endomysium (Robertson, 1957) . The myofibrils are bathed by the sarcoplasm, which contains soluble substances, mitochondria, nuclei, and a complex of internal membranes (Bloom and Fawcett, 1968) . Porter (1961) described the complex of 3 of internal membranes as consisting of two main systems: (1) the sarco- plasmic reticulum, which is a branching network of thin, irregularly shaped vesicles feund between and enveloping the fibrils; and (2) the T-system, a set of transverse tubules about 0-3F in diameter, which ori- ginates at or near the sarcolenma and projects inward into the muscle fibers. He further stated that the sarcoplasmic reticulum seems to func- tion in controlling the relaxation process. The myofibrils have been described-as the contractile elements of striated.muscle by Huxley (1960, 1965, 1967). He stated that myofibrils measure l-Zp across and run parallel to the long axis of the fiber. He fUrther stated that the cross striations are due to regularly arranged myofilaments which are composed of the three principal myofibrillar proteins, myosin, actin, and tropomyosin. Bloom and Fawcett (1968) listed and described the main bands of the myofibril which are summarized below: (1) The A band contains myosin filaments measuring about 100A.x 1.5“. ‘When viewed under the polarizing microscope, the A.band appears bright or anisotropic. On the other hand, it stains dark with iron- hematoxylin. . (2) The I band contains actin filaments, which are 50A thick and extend about In from each side of the 2 line. These filaments, which may also contain some tropomyosin, are often called "thin” filaments in contrast with the thicker myosin filaments in the A.band. The thin fila- ments appear dark or isotropic under the polarizing microsc0pe and remain essentially unstained with iron-hematoxylin. (3) The 2 line contains tropomyosin and runs across the myofibril to bisect the I band. The Z line is visible with dark phase contrast microscopy and appears dark with many stains. One sarcomere (the 5 distance in which the striation pattern of the myofibril repeats itself) is defined as the distance between two successive Z lines. (4) TheMline runs across the middle of the A band, bisecting it and apparently holding the thick filaments together at their midpoint. The clarity of this line varies with the degree of contraction of the myofibril and with the method of preparing the histological section. Thus, at times it is hardly detectable. According to Huxley (1960, 1965, 1967), a cross section of the myofibril taken in the region of overlap between the myosin and actin filaments shows each myoSin filament to be surrounded by six actin fila- ments in hexagonal array. Each actin filament is in turn shared by the six neighboring myosin filaments. He fUrther showed that each thick fila- ment has distributed along its length many short lateral prOjections or cross-bridges. These appear to extend outward and touch the thin fila- ments. Huxley (1960, 1965, 1967) postulated that the cross bridges inter- act with the thin filaments, and that the myosin-actin interaction corres- ponds to the fermation of actomyosin during muscle contraction. Proteins of the Myofibril .All but a small percentage of myofibrillar material has been feund to be protein (Perry, 1951; Perry, 1967b), and consists mainly (80-90%) of myosin, actin and tropomyosin (Poglazov, 1966; Perry, 1967b). The contribution Of the various myofibrillar proteins as reported by different investigators is summarized in the fellowing table: a g . - . - The Protein Composition of the Wofibril Percent of total myofibrillar protein 1966 1965 Perry (1967b) recently pointed out that myofibrillar proteins interact strongly with each other, thus during purification one gets incomplete extraction and persistent impurities. Consequently, he has indicated that composition data for the myofibril is only approximate. He has also listed several minor components of the myofibril, which have not yet been well defined. In addition to the myofibrillar proteins listed by Ebashi (1966), Perry (1967b) also included the inhibitory factor, fibrillin, and ribonucleoprotein. Other proteins have been found in the myofibril, which have different properties than those mentioned by Perry (1967b) and Ebashi (1966). Poglazlov (1966) lists them as contractin, Tmyosin, metamyosin, Y protein, and Aprotein. Although these constituents have certain dis- tinguishing features, they are similar in many respects, and probably consist of complexes of the already known proteins (Poglazov, 1966) .' This agrees with Ebashi (1966), who stated that the well-defined myo- fibrillar proteins make up 9695 Of the total. Myosin: I Extraction of myosin has been reviewed by Huxley (1960) , who stated that two solutions have conmonly been used for extracting myosin. 1) The Cuba-Straub solution, which consists of 0.3 M KCl and 0.15 M phosphate 7 buffer at pH 6.5 (Cuba and Straub, 1943) , usually extracts some actin along with myosin. Its effectiveness can be increased by adding 5 x 104 M ATP (Huxley, 1960). 2) The Hasselbach-Schneider solution, which is composed of 0.47 M KCl, 0.1 M phosphate buffer, and 0.01 M sodium pyro- phosphate at pH 6.5 (Hasselbach and Schneider, 1951) , extracts myosin with almost no contamination from actin. Its effectiveness can be increased by adding 10'3 M MgClZ (Huxley, 1960). Huxley and Hanson (1957) have developed a solution which gives excellent selective extraction of myosin. It consists of 0.6 M KCl, 0.1 M phosphate buffer, 0.01 M sodium pyrophosphate and 10'3 M MgCl2 at pH 6.5. According to Huxley (1960) and Bendall (1964), extracted myosin remains soluble if the ionic strength is lowered to 0.2-0.3. Thus, removal of contaminating actin can be achieved by adjusting the ionic strength to 0.3, which precipitates any actomyosin present (Bendall, 1964). Similarly, myosin can be separated by adjusting the ionic 3 M ATP, thus superprecipitating the acto- strength to 0.15 and adding 10' myosin (Huxley, 1960). By lowering the ionic strength to 0.05, the myosin can be precipitated, leaving the more soluble proteins in solution (Huxley, 1960). The widely used method of Perry (1955) for purification of myosin utilizes the above technique to eliminate impurities, however, myosin prepared in this maImer is far from pure (Perry, 1967a) . Richards 9; 31. (1967) pointed out that myosin preparations have been plagued by the presence of myokinase, AMP deaminase, micleic acids, myosin aggregates, myosin-nucleic acid canplexes, unidentified proteins, and decreased ATPase activity. They indicated that attempts to purify myosin have all yielded impure preparations or reducedmyosin ATPase activity. Thus, they outlined a procedure using DEAE Sephadex A— 50 for obtaining monomeric myosin essentially free of contaminating substances and having high ATPase activity. They reported that myokinase was the only contaminant, and that 50% of the original myokinase activity could be removed during purification. Myosin has been studied extensively (Poglazov, 1966) , yet much confusion persists about the size of the myosin monomer (Dreizen, 1967) . Perry(1967a) eiqalained that the tendency of myosin to aggregate as well as difficulty in purification have caused errors in determining its molecular weight. Dreizen (1967) , in reviewing molecular weight studies on myosin, indicated that reported values have ranged from 420,000 - 1,500,000, but stated that most workers now agree on a value of approxi- mately 500,000. Richards gt gl_. (1967) have also reported a molecular weight for highly purified myosin of about 500,000. After reviewing measurements on the size of the myosin molecule, Perry (1967a) stated that it is about 1550 A long, from 200 - 400 A in width at the head and about 20 A wide along the tail. Some workers have attempted to simplify the myosin molecule by breaking it down into subunits (Poglazov, 1966). Perry (1967a) pointed out that the only real subunits of the myosin molecule are those obtained by using dissociating agents. He stated that the subunits usually have a molecular weightof 160,000 - 260,000 and have never, been known to exhibit biological activity. According to Perry (1967a) , the myosin molecule has also been split up by controlled proteolytic digestion to yield "fragments" of the myosin molecule. He stated that light mero- myosin (LNM) and heavy meromyosin (HIM) are fragments rather than subunits "since they are produced by the breaking of peptide bonds and do not pre- exist as units from which the molecule is made up". HIM has a molecular weight of 380,000, is more soluble than myosin, contains the ATPase activity and the actin-combining ability of myosin (Perry, 1967a; Poglazov, 1966) . LAM has a molecularweight of 120,000, retains similar solubility pro- perties to myosin, has no known biological activity, and seems to be only structural in function (Perry, 1967a; Poglazov, 1966). I According to Bendall (1964) , the isoelectric point of myosin in 1(Cl solutions is 5.4, however, on addition of Mg“ or Ca” ions it increases to 9.3 due to the unusual affinity of myosin for divalent ions. Lawrie (1966) stated that the affinity for divalent ions is due to a high content of glutamic, aspartic, and the dibasic amino acids. Poglazov (1966) stated that the two most important chemical pro- perties of myosin are its ATPase activity, and its ability to combine with actin. Bendall (1964) stated that myosin ATPase is activated by Ca” , inhibited by Mg” , and has two pH optima (pH 6.4 and 9.3). He also stated that myosin ATPase is very heat and acid sensitive; its activity is influenced by sulfhydryl groups in the molecule; and its behavior is modified by the presence or absence of actin. Bendall (1964) pointed out that since myosin complexes with actin to form actomyosin, the term "actin-modified ATPase" really refers to actomyosin ATPase. He also stated that the behavior of actomyosin ATPase depends on the ionic strength of the medium. At high ionic strength (0.6MKC1), the addition of ATP causes dissociation of actomyosin so that its ATPase now behaves much like myosin ATPase, i.e. , it is activated by Ca” and inhibited by Mg”. At low ionic strength (0.15M KCl) , the addition of ATP superprecipitates actomyosin, causing it to "cmtract".1i;sddinen$ion— ally. In the superprecipitated state, actomyosin ATPase is activated by Mg" if trace amounts of CaW are present. 10 The localization of myosin in the sarcomere has been reviewed by Huxley (1960). He stated that Hasselbach (1953) and Hanson and.Huxley (1953, 1957) used high ionic strength solutions of KCl, sodium pyro- phosphate and MkCl2 to study the localization of myosin in the myofibril. Since such solutions remove the A band completely, these authors suggest that myosin is located in the A band. Seifter and Gallop (1966) reviewed.the use of antibodies in localizing myosin. Since antisera prepared against myosin generally precipitate at the A.band (Seifter and Gallop, 1966), antibody studies further confirm localization of myosin at this point. Actin Actin is firmly attached to the muscle structure, so that severe treatments are needed to extract it (Huxley, 1960), i.e., acetone‘treat- ment (sum-16,1942) or 0.6 M KI (A.G. Szent Gybrgyi, 1951b). Treatment with-0.6 M KI'has been found to slowly denature actin (Lewis e3 £11., 1963) . The extraction method of Straub (1942), or variations thereof, has been the starting point for most actin preparations (Bendall, 1964)., Prepar- ation involves: l) pre-extraction of myosin from the muscle; 2) acetone treatment to denature various remaining proteins and to.free the actin from the muscle structure as well as removal of the lipids; and 3) ex- traction with neutral distilled water to obtain globular actin (G-actin). .Actin can exist in two ferms, globular (G-actin) and fibrous (F-actin) Bendall, 1964). In vivo it is believed to exist as F-actin (Huxley, 1960). According to Briskey (1967), the only successfhl attempt in isolating F-actin directly was carried out by Hama.et.§1, (1965). They began by pre-extracting myosin from the muscle, then perfOrmed short, mild tryptic digestion on the residue to release F-actin from.the muscle structure . 11 Once extracted, actin has usually been purified by repeated poly- merization-depolymerization (Adelstein SE 21., 1963). Conventional techniques, however, have failed to purify actin sufficiently for studying its molecular parameters (Hayashi, 1967). According to Hayashi (1967), tropomyosin and myokinase are probably the most persistent impurities found in actin preparations. He further stated that extraction of actin at 0°C reduces the amount of tropomyosin, yet even under these conditions enough tropomyosin remains to alter the properties of actin. Ebashi (1966) pointed out that actin extracted.from.acetone powder (Straub-type actin) usually containscx-actinin as an impurity. Ebashi and.Ebashi (1965) were able to remove essentially all of thecK-actinin by raising the KCl concentration to 3.3 M. They concluded that this treatment precipitatesCX-actinin and leaves actin in solution. (Z-actinin is present in KI-extracted actin solutions, but seems to be absent in most Straub-type actin preparations (Maruyama, 1965 a, b). Troponin, although quite instable to acetone treatment, is present in some actin preparations (Ebashi and Ebashi, 1964; Briskey, 1967). Tropomyosin,C*-actinin, and B-actinin all interact with actin to change its behavior (Hayashi, 1967; Maruyama and Ebashi, 1965; and Maruyama, 1965 a, b), thus it is important to remove these contaminants before studying the properties of actin. Adelstein SENS}: (1963) purified actin on a column of Sephadex G-200 to obtain actin free of smaller mole- cules and certain enzymes. They made no statement concerning the amount of tropomyosin or other impurities commonly feund in actin preparations. Seraydarian et a1. (1967) obtained so-called "pure" actin by extraction at 0°C, treatment with 3.3 M KCl and other modifications. The molecular weight of the G-actin monomer is not known with certainty due to difficulty in obtaining purified actin, and the tendency 12 of actin to polymerize (Huxley, 1960; Hayashi, 1967). In feur recent reviews (Bendall, 1964; Poglazov, 1966; Briskey, 1967; and.Hayashi, 1967), values from 50,000 to 150,000 were cited for the molecular weight of ‘ G-actin, with most of the values falling between 56,000-70,000; (On the other hand,there is no agreement on the molecular weight ofrFeactin since it behaves as either a polymer or a dimer (Huxley, 1960; Poglazov, 1966; and Briskey, 1967). Briskey (1967) stated that the G-actin molecule measures approx- imately 55A in diameter. He described F-actin.as a double.helical polymer of G-actin monomers, making a complete turn every 700.A and measuring 80A in diameter;‘ The.actin molecule has 450 amino acid residues (Laki and Standaert, 1960), with large amounts of glutamic and aspartic acids (Carsten; 1963). According taiBriskey (1967), actin has more proline than most proteins. He suggestSfthat'the proline content may account for the low percentage of helical structure (30%) as compared to myosin (56%) and tropomyosin (96%). According to Poglazov (1966), the two most important properties of actin.are'the.G~F transformation and the interaction between actin and.myosin. The latter has already been discussed. Hayashi (1967) has represented the G-F transformation by the fellowing equation: . n G-ATP M F-ADP + nPi ‘ where: G-ATP is G-actin containing ATP, FsADP is F-actin containing.ADP and Pi is inorganic phosphate. Poglazov (1966), in reviewing the G-F transfermation, stated that G-actin is converted to F-actin, when the ionic strength of the medium is raised to 0.01 - 0.15. The change is enhanced by Mg‘Iand.is inhibited by Ca**in the presence of monovalent ions. He 13 further stated that polymerization likely occurs due to hydrogen bonding' between sulfhydryl and amino groups and between hydroxyl and amino groups. ATP also.seems to be involved in polymerization (Poglazov, 1966). Recently,.G+aetinfhas been.fbund to polymerize in the absence of nucleo- tides and divalent ions (Kasai e; g1., 1964; Hayashi, 1967)."Thus, theories on the role of the G-F transformation in muscle contraction should bare-evaluated (Mmmaerts, 1966; Hayashi, 1967).. The localization of actin in the myofibril was studied by Rozsa st 31. (1950), who reported that synthetic actin fibers.resemb1e'the‘ thin filaments of the I band when viewed.under the electron microscope. Hence, A.G.,Sent286y5rgyi (1951a) concluded that the I band contained‘ actin. This was later confirmed by Hanson and.Huxley (1955);'wh0'used 0.6M KI to extract actin from myofibrils containing no A band‘or M line. The thin filaments were removed by this treatment. Szentkiralyi (1961) prepared HMM5=then‘added this to isolated myofibrils and observed‘that‘ the I band became darker in appearance as the HIM became attached‘to the actin filaments;‘ This further confirms the presence of actin filaments in the I band;" EndO‘e£;§13‘(1960) used the fluorescent antibody technique to show that tropomyosin and tr0ponin are capable of complexing with the material in the A.band. Seifter and Gallop (1966) later.reviewed the use of anti- bodies in localizing actin,.and suggested such studies.indicated actin may be present in both the A and I bands. However,.they concluded that: caution must be used in interpreting results obtained with antibody tech- niques, especially since impurities in the antigens alter the antibodies. 14 Tropomyosin B Tropomyosin was first isolated and studied by Bailey (1946, 1948) . Tropomyosin prepared by his method is usually called tropomyosin B to distinguish it from paramyosin (tropomyosin A), which is found in certain muscles capable of prolonged tetanic contraction (Poglazov, 1966; Seifter and Gallop, 1966). The method of Bailey (1948) involves : 1) ex- traction .of muscle with distilled water; 2) treatment with organic solvents (ethanol followed by ether) to remove the lipids and denature the unwanted components; 3) extraction of tropomyosin B with 1M KCl; and 4) purifi- cation by repeated isoelectric precipitation and anmonium sulfate fraction- ation. Tropomyosin B has been reported to contain some impurities. For example, itis often isolated as a complex containing nucleic acids, which are difficult to remove (Hamoir, 1951; Needham and Williams, 1963; Carstens, .1968) . Other contaminants found in tropomyosin B preparations include: actomyosin (Hamir and Laszt, 1962), tryptophan, which is not present in tropomyosin B (Kominz _e_’g 31. , 1954), and up to 5% of low molecular weight material (Woods, 1967) . On using free boundary electro- phoresis Davey and Gilbert (1968) found tropomyosin B to be about 70% pure, but were able to achieve further purification using DEAE-cellulose chromatography. Tropomyosin B preparations usually do not contain troponin unless prepared without the use of organic solvents (Ebashi, 1963; Perry 1967a). However, tropomyosin B prepared in the presence of a sulfhydryl reducing agent or EDTA has been shown to have troponin activity (Mieller, 1966) , or a tendency to aggregate (Woods, 1967) , which also indicates troponin contamination (Ebashi and Kodama, 1965). 15 As with other myofibrillar proteins, there is uncertainty regarding the molecular weight of tropomyosin B. Although reportedwvalues range between 53,000 - 140,000, until recently the generally accepted molecular weight has been 54,000 (Seifter and Gallop, 1966; Poglazov,.1966). How- ever, evidence now indicates that the molecular weight of tropomyosin B may be around 70,000 (Holtzer‘et_gi,, 1965; WOods, 1965, 1967). Further- more, tropomyosin B can be dissociated into two identical subunits with a molecular weight of 34,000, which points to a molecular weight of approximately 68,000 fer tropomyosin B (WOods, 1965, 1967).. The molecule is.believed to measure 340-385A in length and 14A in diameter (Tsao e_t__§1_., 1951). Due to its low content (16%) of glycine, alanine, and.serine residues, as well as a high content (40%) of amino acids with free acidic or basic groups, tropomyosin B contains the highest zwitterion charge density of any known protein (Seifter and Gallop, 1966). Tropomyosin B has two free sulfhydryl groups and a very low proline content, which is consistent withfits high percentage oftX-helix (Seifter and Gallop, 1966). Tropomyosin thas no free N-terminal groups (Bailey, 1951) and the poly— peptide chain may be folded back on itself (Huxley, 1960; Poglazov, 1966). Poglazov (1966) stated that tropomyosin B has the fellowing proper- ties: 1) It is soluble in distilled water and dilute salt solutions at all pH values outside the range of 4.5 — 6.5. 2) It shows a considerable increase in viscosity below 0.01 M KCl. 3) It has an isoelectric pOint of 5.1. 4) It resists denaturation by heat, acid and organic solvents, while urea and surfactants have only a slight effect. TrOpomyosin B has no known enzymatic properties and does not combine with myosin (Bendall, 1964). Further, it has no effect on actomyosin 16 (Ebashi, 1966), but does, however, combine with actin (Hayashi, 1967). The amount of protein in the I band exceeds the amount of extract- able actin (Hanson and.Huxley, 1957; Huxley and Hanson, 1957); ‘This residual protein is thought to be in.part tropomyosin B (Huxley, 1953; Huxley andeanson, 1957). Electron microscope studies have shown that the residual material in the Z and I bands has a crystal lattice similar to that of*tropomyosin B (Huxley, 1963; Knappeis and Carlsen, 1962; Cohen, 1966). Furthermore, the thin filaments appear to be attached to the 2 band by‘a network of tropomyosin (Huxley, 1957; Knappeisend‘ Carlsen, 1962);" Thus, tropomyosin appears to be localized mostly in the I band with assmall.amount in the 2 line (Hansen and Lowy, 1963;'1964). The presence of trepomyosin B, as well as troponin, in the I.band has recently been confirmed by antibody techniques (Endo e£_§1,, 1966; Pepe, 1966). Troponin Troponin has been called the ESTA-sensitizing factor (ESF) by Perry (1967a) and the relaxing Irotein by Natanabe and Staphrans (1966). The discovery and description of trOponin has been described by Perry (1967a) and.Ebashi (1966). They indicated that natural actomyosin is usually relaxed by calcium chelators, whereas, synthetic actomyosin is often unaffected by the presence or absence of Ca". Ebashi (1963) dis- covered a factor, which would restore the ability of synthetic actomyosin to relax in the absence of CaII. His preparation resembled tropomyosin B, but was subsequently shown to be a complex of tropomyosin B pluS' another factor, which he called troponin (Ebashi and Kodama, 1965,' 1966). Simultaneously, A. Szent-Gydrgyi and Kaminer (1963) isolated a preparation,that they called metin. This was subsequently shown to be a 17 complex of tropomyosin B and trOponin (Azuma and watanabe, 1965 a, b), and is closely related to the preparation isolated by Ebashi (1963). Perry (1967a) has stated. that all ESF preparations-except that ' prepared.bnyer y et_§1, (1966) have contained tropomyosin as the major component. On attempting to isolate ESF from."extra protein" by chromo- tography on DEAEecellulose, Perry 33 gl, (1966) detected ESF activity in several fractions*throughout the chromatogram. The reason.ESF'iS'not‘ cleanly separated from other components is not apparent. (Perry 33 313, 1966). The properties of troponin have not yet been well described (Perry, 1967a). eTroponin'isra globular protein, which will complex with'tIOpo- myosin B, thu3‘increasing the tendency of tropomyosin B to aggregate‘ (Ebashi,-1966);" The aggregated complex is called.native tropomyosin and has a higher viscosity, greater flow birefrigence and a larger sedimené tation constant than does tropomyosin B (Ebashi and Kodama, 1965). There is a relationship between ESF activity, the E278/B260 ratio, and the thiol content of tropomyosin (Perry, 1967a). Perry (1967a) indicated that little is known regarding the mode of action of ESP; ‘HOwever, it affects the Mg**activated actomyosinrATPase but not the CaTIactivated myosin ATPase. EbaShi (1966) suggested that‘ troponin influences the actin.moiety of actomyosin. In the same investi- gations, he presented evidence that trOponin can not act alone, but.requires tropomyosin B. .On the other hand, Perry 33 31.,(1966) and Perry (1967a) suggested that troponin may not require tropomyosin B. Endo 35,21, (1966) used antibody techniques to localize native' tropomyosin in the myofibril. Results suggested that trepomyosin’B and troponin are both distributed along the entire length of the thin filaments. 18 According to Peachey (1968) , two roles are thus implied for tropomyosin: l) as a structural part of the thin filaments, and 2) as a means of con- trolling the interaction of actin by the presence or absence of Ca”. Oi-actinin Ebashi and Ebashi (1964, 1965) first isolated and studiedd-actinin. Since this protein complexes with actin (Maruyama and Ebashi, 1965'; Briskey _e_1_:_ §_1_. , 1967b) , the contaminating actin must be removed from all purified preparations. Ebashi and Ebashi (1965) and Ebashi and Maruyama (1965) found that ok-actinin could be precipitated from the contaminating actin by treatment with 3.3 M KCl. Seraydarian e_t_ §_l_. (1967)prepared purified Oi-actinin by employing the method of Ebashi and Ebashi (1965) and Ebashi and Maruyama (1965) along with their own modifications. Their method involved: 1) extraction of myosin from muscle with a KCl-phosphate solution; 2) washing of the muscle in a low ionic strength medium; 3) extraction of cat-actinin with a low ionic strength medium for several hours at 20°C; 4) partial purification by ammonium sulfate fractionation; and 5) final purification by precipitating the (x-actinin with 3.3 M KCl. Preparations of OK-actinin obtained by amonium sulfate and potass- ium chloride fractionation have been found to contain 3 components, with sedimentation constants of about 68, 108 and 258 (Ebashi, 1966). According to Ebashi (1966) all three components have the same physio- logical activity. He concluded that the 253 component is probably an artifact formed during preparation, while either the 68 or the 108 com- ponent is found in the myofibril. The amino acid composition of IX-actinin resembles that of actin, thus it has been suspected of being denatured actin (Ebashi, 1966) . Purified OI-actinin does not complex with myosin, but forms a complex with l9 F-actin (Maruyama and Ebashi, 1965; Briskey et al., 1967b). If Oi-actinin is added to a suspension of F-actin under proper conditions, it will form a gel (Ebashi, 1966). Ebashi (1966) pointed out that the presence of IX-actinin is known to enhance the superprecipitation of actomyosin. Thus, he postulated that Oi-actinin plays a direct role in muscle contraction. On the other hand, Briskey et al. (1967a, b) presented evidence that the Oi-actinin-actin interaction may be unrelated to muscle contraction. Goll _e_’_c_ a_l_. (1967) were able to detect c><-actinin in the 2 band by a tryptic digestion and subsequent treatment of the solublized fration with 3.3 M KCl. They concluded that (Di-actinin is at least partly located in the Z band. Similarly, Ebashi (1966) presented preliminary results of antibody studies, which indicated that ot-actinin is located in the 2 band, and perhaps in the M band. F-actinin Maruyama (1965a,.b) isolated a protein factor which inhibited net- work formation in Straub-type F-actin and restricted the fiber length to 1-2”. He named the factor B-actinin and prepared it as follows (Maruyama et'al., 1965; Maruyama, 1965a, b): l) Myofibrils were prepared according to theimethod of Perry and Zydowo (1959a) . 2) Myosin was extracted from the myofibrils with a solution cf KCl and potassium phosphate. 3) Actin was extracted with a solution of KI. 4) B-actinin was separated from actin by amnonium sulfate fractionation. According to Maruyama (1965b) , P-actinin prepared by the above method has an amino acid composition similar to that of actin, and a molecular weight of 300,000 in 0.1 M KCl. Maruyama (1965b) also stated that fl-actinin restricts the fiber length of F-actin _i_n vitro to that of 20 F-actin filaments in vivo (l'Zfi). Thus, Ebashi (1966) suggests that B-actinin may function in muscle development rather than in muscle contraction. Inhibitory Factor Hartshorne 9.1}. 31; (1966) isolated a factor from skeletal muscle, which they called the inhibitory factor (IF). This factor, in the absence of EGTA, inhibits the MgH-activated ATPase of desensitized actomyosin (natural actomyosin from which ESF has been removed). IF is probably a component of the myofibril (Perry, 1967b). Hartshome 31331. (1967) prepared IF by extracting myofibrils with a high ionic strength solution at pH 8.6, then lowering the ionic strength to precipitate the salt-soluble components, and finally isolating IF from the supernatant by armnonium sulfate fractionation. IF prepared in this manner appears to be protein in nature, since it is precipitated by amnonium sulfate, destroyed by trypsin and heat, and loses activity on prolonged storage at 0°C (Hartshorne _e_t_ §_1_., 1966). The significance of IF is not yet clear (Hartshorne et 31. , 1966). It is often found associated with ESF activity, and sometimes a decrease in ESF activity is accompanied by a rise in IF activity (Perry, 1967a). However, the low levels of tropomyosin in IF preparations, as well as differences in their properties suggest that the two factors may not be the same (Hartshorne e; 31. , 1966). Extra Protein Grog On extracting myosin with high ionic strength salt solutions and ATP, A.G. Szent-Gydrgyi _e_1_:_§_l_. (1955) found that other proteins are also solublized. On lowering the ionic strength to about 0.05, the myosin precipitated, leaving the so-called "extra protein" in solution (Poglazov, 1966). The extra protein fraction is heterogeneous (Perry 8 Zydowo, 1959a) and probably accounts for about 7% of the total myofibrillar protein. 21 Perry and Zydowo (1959a) separated extra protein into four com- ponents by means of DEAE-cellulose chromatography. Fraction I was shown to consist mostly of sarc0plasmic components, which could.not be removed from the myofibrils by extensive washing. Fraction II was feund to consist of a water-soluble and a water-insoluble component, and has not been identified. Fraction III consisted of tropomyosin B plus some other protein. Fraction IV contained considerable bound ribonucleic acid. Perry and Zydowo (1959b) characterized the ribonucleoprOtein further, and postulated that it may be associated with protein synthesis. Nature of the Actomyosin Complex Composition. Perry (1967a) stated that natural actomyosin, which is extracted and purified directly from.musc1e, is different from synthetic actomyosin, which is prepared from purified actin and myosin". He indicated that natural actomyosin is usually relaxed by calcium chelators, whereas, synthetic actomyosin is not similarly relaxed. .It has been shown that minute amounts of CaH'regulate the contraction-relaxation cycle, thus synthetic actomyosin may not represent the complete fundamental system of muscle contraction (Ebashi, 1966). The discovery of several new myofibrillar proteins (Ebashi and Ebashi, 1964, 1965; Maruyama, 1965 a,b) and of their effect on actomyosin suggested that natural actomyosin may contain.other proteins besides actin and myosin (Ebashi, 1966). Briskey (1967) stated that natural acto- myosin probably contains myosin, actin, tropomyosin, oeactinin, B-actinin and troponin, as well as other unknown proteins. Effect of ATP and EDEA~ On the basis of viscosity, light scattering, and sedimentation in 22 the ultracentrifuge, several workers (A. Weber,.1956; Barany and Jaisle, 1960) have shown that ATP appears to cause.dissociation of actomyosin at high concentrations (0.6M) of KCl (H.H. Weber, 1964') .' Under suitable conditions, ATP is rapidly broken down by actomyosin ATPase, so that the viscosity and light scattering soon return to their fermer values (Bendall, 1964). Pyrophosphate in the-presence~oergf’seems to dissociate acto- myosin in the same manner as ATP (Azzone and Dobrilla, 1964), except that pyrophosphate is not hydrolyzed by actomyosin ATPase.- Actomyosin, there- fore, cannot eliminate the pyrophosphate and remains dissociated (H.H. weber, 1964; Bendall, 1964). I‘Little is known about the mechanism by which ATP and its analogs affect actomyosin (Azzone and Dobrilla, 1964),.but according to H. H. . ‘Weber (1964) it is likely not a simple dissociation of.actin and.myosin. .Although the majority of workers have concluded that ATP dissociates actomyosin (Johnson and.Rowe, 1964), some have suggested that actomyosin undergoes shape changes without dissociatingu(Blum:and Morales, 1953; Morales 3131., 1955; von Hippel 31 31., 1957). . Johnson and Rowe (1964) presented evidence Showing that ATP may induce several changes in the actomyosin molecule. They pointed out that.if,the.simple dissociation theory is correct, analytical ultracentrifugation.of actomyosin in the presence of ATP should produce two peaks.corresponding to F-actin and myosin. Instead, a "slow diffuse" peak appeared as well as a "myosin- like" peak,.which seemed to contain varying amountS'of'Gractin. Thus, they concluded.that the action of.ATP on actomyosin is mere than a simple dissociation. According. to Perry (1967a), the ATPase. and. actin-combining pro- perties of myosin are probably controlled through separate active centers, although the mechanism by which ATP influences the two separate centers 23 is not clear. .By studying the effect of EDTA on individual glycerinated.musc1e fibers, Watanabe and Sleator (1957) showed that BETA is capable of relax- ing contracted fibers. Subsequently, weiner.and.Pearson (1966) feund that a lethal intravenous injection of EDTA-in1rabbit5‘inhibited the post-mortem shortening and the inextensibility associated with develop- ment of rigor mortis. Similarly, H. H. Weber (1964) listed EDTA.as an inhibitor of the myosin-actin interaction. On.the other hand, Azzone and Dobrilla (1964) stated that EDTA does not dissociate the actomyosin complex as does ATP and its analogs. The effect of EDTA on actomyosin likely.results from the chelation of metal ions, as EDTA has been shown not to interact'with actomyosin (H. H. weber, 1964). Furthermore, the inhibitory effect of EDTA is abolished on adding excess Ga”'to the system (H.H. weber, 1964). Bacterial Action on Meat Proteins Frazier (1958) indicated that temperature is the most important factor in determining the type of microorganisms that will grow, and thus determines the type of spoilage. He further stated that psychrophiles are favored under prOper refrigeration. According to Evans and Niven (1960), the type of bacteria accumulating un.fresh.meat:stored at 10°C or below are various strains of Pseudomonas, Achromobacter, Lactobacillus, iMicrobacterium and Micrococcus. On the other hand, meSOphiles such as coliform.bacteria and.species of Bacillus and Clostridium will proliferate at intermediate temperatures (Frazier, 1958). I Early workers assumed proteolysis to be a.major process in the bacterial spoilage of beef, poultry, and fish.(Lerke.3£“31,, 1967). Jay (1966, 1967) and Jay and Kontou (1967) showed that the ability of certain 24 bacteria to spoil meat is not related to their proteolytic activity, but the primary proteins of meat are attacked only in advanced stages of bacterial spoilage. The fact that non-proteolytictstrainS'are capable of spoiling meat would suggest that substances other than proteins are attacked by the bacteria (Jay, 1967; Jay and Kontou, 1967).‘ Jay and Kontou (1967) investigated.possible.sources of bacterial nutrients and suggested that the low molecular weight compounds in the sarcoplasm may be used by bacteria as sources of.nitrogen. On this basis, Jay and Kontou (1967) tested fer disappearance.of free amino acids and nucleotides during bacterial growth. They fbund that the amounts and type of amino acids and nucleotides decreased during“storage. Lerke 33 31. (1967) studied the bacterial spoilage of fish muscle. By separating the soluble nitrogenous components of fish muscle into protein and.non-protein fractions, they showed that spoilage of fish (as measured by the usual chemical and organoleptic tests) is probably not due to the breakdown of soluble proteins. .Instead; bacterial spoil- age occurred only in the presence of non-protein nitrogen components. EXPERIMENTAL METHOIB Unless otherwise specified, all work was performed at 3°C. Distilled water (was run through a Barnstead mixed bed ion exchanger before use. Solutions were prepared at room temperature and final pH adjustments. were made at 3-5°C. Reagent grade chemicals were used unless otherwise stated. Cellulose ion exchangers utilized were washed with 0.5 N NaOH, briefly with 0.5 N HCl, again with. 0.5 N NaOH, and distilled water before packing into columns (Sober and Peterson, 1962) . Aminoethyl-cellulose was washed with 0.1 N NaOH, 0.1 N HCl, 0.1 N NaOH, and water. Cellulose columns were packed under nitrogen. pressure beginning at 0 psi and. reaching. a maximum of 10 psi at the completion of the cOlumn. The column were equilibrated with buffer in the .cold before use. Gel filtration media were allowed to swell in water or eluting buffer before packing into columns. Gel columns then were equilibrated with eluting buffer and tested for uniformity with Blue Dextran before . using. Column void volumes were also determined using Blue. Dextran. . Centrifugations are described in terms of the relative centrifugal force developed at the tip of the centrifuge tube. Sample Preparation Rabbit Loflngissimus Dorsi Female rabbits (3-5 lbs.) were obtained locally, killed by exsan- guination, and transferred inmediately to a cold room at 2‘-4°C. The left longissimus dorsi muscle was removed, trinmed free of connective 25 26 tissue and the desired amount of muscle was weighed. Removal of Sarcoplasmic Fraction The muscle was homogenized with 12 volumes of a solution containing 0.25 M Sucrose, lmM EDTA and 0.05 M tris (hydroxymethyl) amino methane (Tris buffer) at (pH 7.6 (Czok and Bucher, 1960; 6011 and Robson, 1967). After standing 10 minutes, the. slurry was centrifuged for 15 minutes. at 20,000 x g. The supernatant was discarded and the residue was washed a second time. The remainingematerial, which is referred toas the washed muscle residue, was used in preparation of myofibrils or myofibrillar proteins. Myofibrils Myofibrils were prepared according to the method of Perry and Zydowo (1959a) . The washed muscle residue was suspended in 9 volumes (based on original weight of muscle) of 0.1 M KCl containing 0.039 M sodium borate buffer, pH 7.1, then centrifuged 15 minutes at 600 x g. The supernatant was discarded and the residue was washed again. The loose upper layer of sedimented material was removed by adding a little KCl-borate solution, gently. swirling and decanting. The firmly SBdi? mented material was discarded, -and the decanted portion was. diluted with KCl-borate solution .to a volume lOtimes that of the original muscle sample. The slurry was spun for 3 minutes at 400 x g. and. the sediment was discarded. The myofibrils remaining in suspension were. washed eight times in KCl-borate-solution, centrifuging each time for 15 minutestat 600 x g. After the lastwashingthe slurry was centrifuged 3 minutesat 400 x g. The myofibrils remaining in. suspensionwere decanted. and con- centrated by centrifugingls. minutes at 600 x g. The sedimented myo- fibrils were stored at 0°C. 27 Salt-Soluble Fraction Perry (1953) reported WebereEdsall solution (0.6 M KCl, 0.04 M NaH003, 0.01 M Nazms) to be a very efficient extractant of myofibrillar proteins. Accordingly, the Weber-Edsall solution was used to extract the total soluble myofibrillar proteins. .. The washed muscle residue was homogenized with 60 ml of weber-Edsall solution per 10 gm of original muscle, or else the prepared.myofibrils were concentrated by centrifuging.fOr 20 mdnutes at 15,000 x g, weighed, and extracted with 45 ml of weber-Edsall solution for every 10 gm of the myofibril preparation. .Extraction was allowed to proceed 20r24.hours,. after which the viscous mass was diluted with 180 ml of weber-Edsall.sol— ution per 10 gm of original .muscleor 135 ml per 10 gm of myofibril prea- . paration. The suspension was.centrifuged for 1 hour at 25,000 x g. The supernatant containing.the.saltrsoluble proteins was saved, and-the residue and loosely-sedimented gel were discarded. The supernatant.was used for preparation of actomyosin, or for studies of salt-soluble proteins. .Actomyosin Actomyosin was prepared.after the method of Morita and Tonomura (1960). Salt—soluble proteins of muscle or of'myofibrils were prepared- as previously described. .The salt-soluble proteins were brought to an ionic strength of 0.2 by addition of 2 volumes of distilled water.. The pH was adjusted to 6.5, and the precipitate fermed was collected by centrifugation fer 15 minutes at 2,000 x g. The supernatant was discarded, and the actomyosin precipitate was dissolved.in 0.6 M KCl at pH 7. The sample was further purified by repeating the precipitation and dissolution cycle twice as described above. The purified actomyosin in 0.6 M KCl was centrifuged 1 hour at 25,000 x g. befOre utilization. 28 Natural Actoryosin Natural actomyosinwas prepared- after the method of Perry and Corsi (1958) with modifications as suggested. by Schaub. _e_t_ 31.. (1967). Salt-soluble proteins. of myofibrils were prepared as previously described. The salt-soluble protein was brought. topH 7.0 with 2 N HCl, and 14001111 of distilledewater (pH 7) were added per 100 ml of protein solution. The precipitate was collected by centrifugation for 15 minutes at 2,000 x g. The supernatant was discarded and the precipitate was dissolved .in 0.6 M KCl (pH 7). Natural actomyosin was precipitated again as described above and was then washed twice with 0.05 M KCl. After each washing, the precipitate was collected by centrifuging 15 minutes at 1,200 x g. The natural actomyosin was dissolved in 0.6 M KCl (pH 7) and character- ized or used for further studies. Individual Myofibrillar Proteins osin Crude myosin was prepared after the method of Perry (1955) . Washed muscle residue was extracted with 30 m1 of a solution of 0.3 M PO and 0.05 M K KCl, 0.10 M KH HPO4 (pH 6.5) per 10 gm of original. 2 4’ 2 muscle. After stirring for 15 minutes, the mixture was centrifuged for. 20 minutes at 20,000 x g. and the sediment was discarded. The volume of the supernatant was measured, and. 14 volumes of distilled waterwere- .~ added with constant stirring. The precipitated myosin was allowed to settle, and the supernatant. discarded. Sufficient KCl was added to bring the ionic strength to. 0.5, after which 0.67 volumes ofwaterwere added to adjust the ionic strength to. 0.3. Centrifugation for20minutes. at 20,000 x g removed any precipitated actomyosin. The ionic strength was adjusted to 0.04 by slow addition (over 10-15 minutes) of distilled water 29 with stirring, whereupon themyosin became insoluble. The precipitate was collected by centrifugation, redissolved by addition of solid KCl to give an ionic strength of 0.5 and reprecipitated by addition. of distilled water to bring the ionic strength. to 0.04. The crude myosinwas dissolved in 0.5 M KCl as before andstored. at. 2.-4°C. for use or further purification. Crude myosinwaspurified. as described by Harris and Suelter (1967) . It was dialyzed at least 24.,hours against. several changesof. 0.2 M KCl containing 0.02 M Tris-HCl. (pH 7.8) . It was then passed through a. combination cellulose phosphate andDEAE-cellulose column, which had been . previously equilibrated with. the same buffer. The combination coltmln consisted of an upper coltmln.(2..5 x10 cm) packed with cellulosephos- 8 phate, coupled in series to a lower column (2.5 x12 cm) packed with .. DEAE-cellulose. The purified myosin was eluted with the same buffer... For purification by the method of Richards 31 31. (1967) , crude myosin was dialyzed 24 hours against. 0.15 M potassium phosphate, pH.-7.5. The myosin was applied to a 1.5.x 7 cm column of IEAE-SephadexA—SO, previously equilibrated. with the same buffer. The purified myosin was then eluted with a linear gradient using 30 ml of 0.15 M potassium phos- phate at pH 7.5. and 30 ml of 0.5 M KCl-0.15 M potassium phosphate, pH 7.5. Actin Actin preparations. were. usually made from the acetone powder of muscle preparedafter the methodof Seraydarian _e_t_:_ 31. (1967)... .The first step .in preparing acetone powder was homogenization of 100 gm of muscle with 330 ml of theGuba-Straub solution (0.3 M KCl - 0.15. M KHZPO4 at pH 6.5), afterwhich. thehomogenate was allowedto. stand..15 minutes. The slurry. was mixed with. 1,330 ml of distilled water, andwas centrifuged for 15 minutes at. 2,000 x g. The supernatant. and those. obtained from subsequent washings were discarded. The residue was .30 washed for 20 minutes with .500 ml of 0.05 M NaHCOS, after which it was centrifuged for .15 minutes at 2,000.-x g. The residue was washed for 10 minutes with 100 ml of. a solutionof 0.05 M NaHCDS and 0.05 M Na2 3, 5 and then was dispersed in..1000.m1 of 5 x 10' M CaClz. After 10 mirmtes, the solid material was.centrifugeddown as before, and.mixedwith 300. .. CO ml of acetone. The mixturewas allowed to. stand forSminutes, andwas .. then recentrifuged under the samenenditions. After two additional- acetone washes, the residue...was spread on a sheet of filter paper. and .. dried at room temperature. The powder was stored in a tightly sealed container for nomore than 2 weeks.-(Monmaerts, 81952). G-Actin was prepared bywthe method of Monmaerts (1952) . , Acetone powder of muscle was dispersed inc30 volumes of distilled water usinga. teflon homogenizer. After extraction for 30 minutes, the viscous mass was centrifuged for 30 minutes at 35,000 x g. The supernatant.was. . saved, KCl was added .. to a concentration of 0.04M, and polymerization. . .. was allowed to proceed for 6. hours. Centrifugation for .21 hours. at..100,000 x g. sedimentedthe. F-actin..,. The supernatant was. discarded and. the..F.-.actin pellet was dissolvedin aminimal amount] of a solution. containinngSmg. ATP (pH 8.2) .per liter. This step usually requ1red stirring. for..2.-hours.. , The polymerization. cycle was. repeated by adding KCl, allowing. poly.-.. merization to proceed, then .collecting. the. F-actin by centrifugation..- The pellets werethen- dispersed ina solution containing 250 mg ATP per liter at pH 8.2.? Dewlymerization. of actin was brought to completion by dialysis for 2 days. against several changes of 1074 M ATP (11118.2). under a nitrogen atmosphere. The. Geactin solution was. centrifuged- 2. . hours at 100,000 x g and stored at 0°C. for characterization. 31 The method of Adelstein e_t_ 31. (1963) was also used to prepare. G-actin. The dried powderwas. extracted with distilled water. as in the method of Monmaerts (1952). After addition of ATP to give a concentration 4 of 5 x 10- M in the G-actin extract,..the actin was passed. through .a. column of Sephadex G-200, which had previously been. equilibratedwith 5 x 10-4 M ATP (pH 8.1). G—actin was eluted with the same buffer- KI-extracted F-actin was. prepared by the methodof Mamyama 33 31 . (1965) . Myofibrils were prepared by the method of Perry and Zydowo . (1959a) , which has been. previously described. Myofibrils were di5persed in three volumes of a solution containing 0.6 M KCl, 0.1 M phosphate buffer (pH 6.4) , 10 NM sodium pyrophosphate and 1 mM MgC12. The mixture. was then centrifuged for lSminutes at 1,500 x g, and the residue was extracted twice more. with. thesame solution. The insoluble residue. was rinsed three. times with. afive-fold volume of 0.5 11M. NaHCOS, andwas then dispersed in three volumes of a solution containing 0.6 M K1, 6 mM sodium thiosulfate, 5 mM B-mercaptoethanol, 1 mM ATP and 30 mM Tris. buffer (pH 7.5) . Extraction wasallowed to proceed for 15 minutes and the slurry was then centrifuged for 30 minutes at 15,000 x g. The super-. natant was collected and dialyzed overnight against a solution containing . 0.1 M KCl, 0.5.mM ATP and. 5 mM Tris buffer (pH 7.5).. Any precipitate. present was removed bycentrifugation. for 10 minutes at 15,000 xg. The . crude F-actin preparation. thus obtained was partially purified by sedi— mentation in the ultracentrifuge. for 2...hours at 100,000 x g.. The super.- natant was saved. for. preparation. of Beactinin. For characterization,..the F-actin pelletwas dispersed in aminimum amount of a solution containing 0.1 M KCl, 0.5 mM ATPand. 5 mM Tris buffer (pH 7.5). The method of Hama _e_t_ 31...(1-965) was used .for. preparation .of Fractin directly from muscle without depolymerization or acetone treatment. .32 Myofibrils were prepared as alreadydescribed. Myofibrils obtained from 10 gm of nmscle were extracted.»-for.e30:minutes with 200 ml of a solution. . of 0.6 M KCl, 0.1 M phosphatebuffer. buffer. (pH 6.4) , 10 11M pyrophosphate and 1 mM MgC12. The insolublematerial was. collected by centrifugation . . . for 15 minutes at 2,000.x.g,. andwasextracted twice again in.thesamew manner- The residue was then. rinsed. in 150ml of 0.05 Mhistidine-HCI . buffer (pH 7.1) ,_ centrifuged. as before, and .re-suspended in 15 ml of- . . histidine-HCI buffer (pH 7.5) at 25°C. As soon as the suspension reached 25°C, 2.5 mg of. trypsinwere addedwith gentle stirring. After 15 . minutes, digestion was. terminated by. addition of 4.0 mg of soybean. trypsin inhibitor. The. solidmaterialwas collected by centrifugation. (15 . minutes at 2,000 x g) andwas rinsed 3-4 times with 0.05 M histidine-HCl buffer as before. In order to. free the F-actin from the muscle structure, the residue was dispersedin 25 .ml..of.0.1 M KCl by means of a. teflon. homogenizer. Centrifugation for 15 minutes at 8,000 x g sedimented the coarse material, leaving the F-actin in the supematant. The F-actin was collected by ultracentrifugation for 2 hours at 100,000 x g, re- suspended in 0.1 M. KCl,.-and if necessary, was clarified by centrifuging. . for 10 minutes at 41,000 x g. The supernatant contained the "natural F-actin". Tropomyosin B Tropomyosin. Bwas preparedaccording to the method. of.Bailey-.(.1948) .. Preparation of . the wsclepowder was performed. at room temperature and . all subsequent steps. at. 241°C. Fresh. rabbit longissimus dorsimuscle was. homogenized with 2 volumes .of water for. l.minute in a Waring. Blender..- After standing 30 minutes, the masswas centrifuged for 15 minutes at 2,000 x g, and the supernatant.was. discarded. The residue was washed with an equal volume of ethanol, then with 4 volumes of ethanol-water (1:1) , twice 33 with 95% ethanol, and then twice with ether, with. centrifuging for 10 minutes at 2,000 x g after .each treatment. The fibrous material was allowed to become semi-dry by evaporation, and was then stored for not more than 2 days in. atightly sealed. container at -20°C. The ether-damp muscle powder was extracted with 10 volumes (w/v) of l M KCl (pH adjustedto 7.0 withwl 1M NaOH) in the cold- After 12 hours, the viscous mixture was centrifuged 20 minutes at 2,000 x. g. The residue was re-extracted with- asmall volume of l M KCl (pH 7.0) , andthe two extracts were combined. The combined extracts were adjusted to pH 4.3 with l N HCl, allowed to stand for -1. hour, and the precipitate was. centrifuged down for 10 minutes at 1,500 .x g. The precipitate was. then dispersed in 5 volumes of distilled water, and the pH was adjusted to 7.0. The total volume was measured and saturated (.NH4) 2804 (containing 1% of freshly added concentrated amonitnn-hydroxide). was slowly added with stirring to give 41% saturation..- Centrifugation for 10 minutes at 1,500 x g removed the precipitate, which was discarded and saturated (NH4) zSO4 - was slowly added to the supernatant with stirring to give 70% saturation. The precipitatedtropomyosin B was centrifuged for 15 minutes at 15,000 x g, then dialyzed ..for 24 hours. against several changes of distilled water. The isoelectric precipitation was repeated at pH 4. 5, and the product was refractionated with ammonium sulfate, saving only the fraction precipitating between 47-70% saturation. . The precipitated tropomyosin , B was subjected to the purification .cycle again and then finallydialized against distilled water to remove the ammonium sulfate. The subsequent. ethanol-ether treatment -for storage of the protein was not .carriedout, but instead the tropomyosianas characterized immediately. Tropomyosin. Bwas also isolated by the method of Mueller (1966) , which was the same as that of Bailey (1948) except that: 1) All reagents 34 after the ether denaturation contained 0.5 mM dithiothreitol. 2) The first extraction with 1 M KCl lasted 15 hours, and the second lasted 3 hours. 3) The protein was purified first by isoelectric precipitation at pH 4.3, a second isoelectric precipitation at pH 4.9, a third iso- electric precipitation at pH 5.2, and dialysis against 0.5m4dithio- threitol. 4) The second purification phase consisted of an amnonium sulfate fractionation, collecting the precipitate formed between 40-55% saturation; then fractionation a second time, saving the tropomyosin B precipitated between 47-55% saturation. Tropomyosin B was also prepared by the method of Bailey (1948) with modifications as suggested by Bodwell (unpublished method) and Woods (1967) . The procedure was actually that of Bailey (1948) with the following modifications; 1) Glass distilled ethanol was used. 2) Reagent grade ammonium sulfate was. recrystallized twice from 10'3 M EDTA by addition of glass-distilled ethanol. 3) All work except denaturation with organic solvents was done at 24°C. 4) All solutions used for ex- traction and fractionation of tropomyosin B, except the saturated ammonium sulfate solution, contained 0.01 M EDTA (pH 7.0) . The saturated ammonium sulfate solution was prepared by saturating 0.2M EDTA .(pH 8.0) with sOlid anmonium sulfate. 5) An extra wash with 1 volume of ethanol-water (1:1) was included as the initial step in denaturation with organic solvents. 6) The purification cycle consisted of. isoelectric precipi- tation at pH 4.4-4.8, centrifugation (15 minutes at 1,500 x g) of the re-dissolved protein, precipitation of tropomyosin B between 41-65% . (NH4) 2504 saturation, isoelectric precipitation at pH 4.4-4.8, preci- pitation of the protein in. the range of 45-65% (M1,) 2804 saturation, and two final precipitations of tropomyosin B between 50-65% (NH4) 2804 saturation. .35 Further attempted purification of tropomyosin B was based on the method of Davey and Gilbert (1968)... Tropomyosin B prepared by the method of Bailey (1948).with modifications utilized by Bodwell (unpublished method) and Woods (1967) was dialyzed for 48 hours against several changes of a solution. containing 0.01 MEDTA. brought to pH 8.2 by addition of solid Tris buffer. The proteinwasthen applied to a 2.5 x 11 cm column of DEAE-cellulose, which had been previously equilibrated with the same . buffer. TrOpomyosin B waseluted with. a linear gradient composed of 100 ml of the starting buffer and 100 ml of the starting buffer containing 1 M KCl. Troponin (ESF) Preparation of ESF by the method of Katz (1966) was initiated by extracting acetone-dried muscle. powder prepared as already. described with 30 volumes (w/v) of 0.1 mM ATP .(pH 7.6) for 1 hour at 25°C. The mixture was centrifuged 20 minutes at 35,000 x g, and the supernatant containing G-actin was saved. The volume of the supernatant was measured, and. 1.1 ml of 0.15 M Tris-nitrate buffer pH 7.6, were added per 10 ml. Solid. KCl and MgCl2 were added with stirring to give final concentrations. of 0.1 M and 0.1 nM, respectively. Polymerization was allowed to proceed . for 16 hours at 2-4°C. The .F-actin. formed was collected after centri- fugation for 2 .1/2 hours at 105,000 x g. The supernatant.was. discarded, . . and the pellet was dispersed. in 15. volumes of 0.1 mM ATP at pH. 7.6 using a teflon homogenizer. Centrifugation of . this suspension. for. 1. hour. at 105,000 x g sedimented theunwanted material and left the G-actin. in solution. Formation of F-actin was. again induced, this time by addition of MgCl2 to a level of. 0.6 mM. This step prevented incorporationof contaminating tropomyosin and ESF into the F-actin polymer. Centrifu- gation for 2 1/2 hours at 105,000 x g removed the unwanted F-actin from 36 solution, after which the supernatant was fractionated by adding saturated- ammonium sulfate solution.containing 1% of freshly added ammonium hydroxe ide. The protein precipitating in the range of 40-70% was collected by centrifugation for 20 minutes at 35,000 x g, and is believed to contain tropomyosin and.ESF. The precipitate was dialyzed against several changes. of distilled water.to remove the ammonium sulfate before characterization. ESF was also prepared by the method of.Azuma.and watanabe (1965b). The supernatant obtained from.the first ammonium sulfate fractionation utilized in the preparation oftX—actinin according to the method.of Seraydarian 3£H31. (1967) was the starting point fer preparing ESF by this method. This supernatant was brought to 55% saturation with ammonium sulfate and was centrifuged for 10 minutes at 10,000 x g. The precipitate obtained was dissolved in distilled water and dialyzed against 0.1 M KCl buffered at pH 7.0 with 0.02 M potassium phosphate. The protein solution. containing ESF was applied to.a.4.5 x 40 cm column of Sephadex G-200, which had previously been equilibrated with the same buffer. ESF emerged.. at the void volume and was stored at 0°C. until removed fer characterization. ohactinin The method of Seraydarian egn31. (1967) was employed fer prepar- ation of" . 2 <3 . ux 0.10 on P d <£ 2 0.05“ L) g d UJ 0.00 ' T ' ' I I I f I ' I 0 100 200 300 400 500 600 EFFLUENT VOLUME - ML. Figure 33. Chromatography of breakthrough peak from Figure 3A on ECTEOLA-cellulose. Sample was applied at 0 m1 effluent volume. Experimental conditions are described in the text. 47 0.15— D. I 2 ‘3 _ U\ 0.10 N P d quu~oe lo I 00:33 no.“ nuance—«vow souuouomoum unequal—sum 530nm mo “—32.33 «0 hues-now .. 0.33. 89 ESF activity has been reported in the following protein preparations; MI‘ (Mleller, 1966), KESF (Katz, 1966), AESF (Azuma and Watanabe, 1965a, b) , and extra protein fractions I, II, and III (Perry e_t_ al., 1966) . As can bee seen in Table I, disc gel electrophoresis of the KESF prepar- ation indicated that it contained only tropomyosin bands (Rm = 0.32 and 0.51). On this basis, ESF appears to be non-protein in nature, or else is simply not separated from tropomyosin by the disc gel system. Another interpretation emerges from a comparison of the disc gel patterns of MI, AESF, and extra protein fractions I, II and III (Table I). These preparations contained common bands at Rm = 0.20, 0.26 and 0.91, suggesting that ESF might be localized in these bands. Since MI was prepared under special conditions to preserve ESF activity, it is interesting to note that the bands at IS“ = 0.20, 0.26 and 0.91 are more intense in MT than in any other tropomyosin preparation. . Furthermore, extra protein fractions I, II and III produce only these bands. The elusive nature of ESF has been noticed in several laboratories (Azuma and Watanabe, 1965 a, b; Perry e_t_ al., 1966; Perry, 1967a). ~The present study suggests that ESF is either non-protein in nature or has an Rm value of 0.20, 0.25 or 0.91 in the disc gel system. The method of Seraydarian gt 11;. (1967) for preparation of ot-actinin appears to yield a fairly pure product. (Table I). Disc gel electro- phoresis of ot-actinin revealed no contamination from actin or tropomyosin. The bands produced by cat-actinin (lgn = 0.14 and 0.18) resemble those produced my myosin (Rm = 0.05-0.10), and the ESF group (Rm .-. 0.20, 0.25 or 0.90). Thus caution must be used in interpreting the significance of bands in this area of the gel separation pattern. 90 B-actinin prepared as described.by Maruyama (1965b) contained tropomyosin (Rm = 0.34 and 0.51), actin (Rm = 0.39), and bands at Rm = 0.25, 0.29 and 0.43 (Table I). The band at Rm = 0.25 has tentatively been identified with the ESF group. Thus, the p-actinin group is tenta- tively assigned Rm values of 0.29 and 0.43. The IF preparation (Table I) appeared to contain some myosin (R In IF preparation contained two bands tentatively identified with the ESF = 0.05 - 0.15) orH :Owuumum awououo muuxo mo aauuooom cauuauoanm .0 0mm can can cam ohm 0mm omm . . . . . _ L 0.0 “do a: .. .35 u 1.3 2m._m><>> 0mm can OHM cam ohm pup-._.b 0mm omm 0.0 H.o m.o ¢.o own an o: cam P ohm _ GAE .Haxcaououo we m.o wcaawwucoa acaua .mcowuuumaoua ewououn we muuooam coHuQMOunm um~0a>uuuH= .3 98m; emu omm h P h 0° 1H.o .m6 1... I . nu W .m.o .e.o I" r Total Extractable Myofibrillar Proteins During extraction of myofibrils with the weber-Edsall solution, 56% of the total myofibrillar nitrogen was solublized. This value is similar to the 58% reported by Hegarty g£_al, (1963), but is considerably lower than the 78-87% reported by Davey and Gilbert (1968) and the 93% reported by Perry (1953). The differences in these values may well be due to variations in the extraction techniques. Since the weber-Edsall extract of the myofibrils is quite viscous, the finer insoluble particles are difficult to remove by centrifugation. In the present work, weber-Edsall extracts were centringed 1 hour at 25,000 x g. On the other hand, Hegarty g£_gl, (1963) utilized centri- ngation fer 1 hour at 1,400 x g, and Perry (1953) for 10 minutes at 10,000 x g. The more severe centrifugation treatment applied in the present research probably sedimented more colloidal material, so that the super- natant contained a lower percentage of the total protein. weber-Bdsall extracts were prepared from six samples of washed muscle residue and from three preparations of myofibrils. Each prepar- ation was analyzed separately using gel filtration, density gradient centrifugation and disc gel electrOphoresis. Gel filtration of weber- Edsall extracts consistently produced 4-5 peaks, but the relative heights of the peaks varied unpredictably. This variation was presumably due to the tendency of myofibrillar proteins to aggregate in solution. A typical gel filtration pattern obtained from the weber-Bdsall extract of'myo-. fibrils is shown in Figure 35. Gel filtration revealed no consistent 93 94 E1 CM AT 254 MU. * J r l o 50 100 150 200 250 EFFLUENT VOLUME - ML. Figure 35. Gel filtration of Weber-Edsall extract. A 2 ml sample of extract containing 1.5 mg protein/ml was applied at 0 m1 effluent volume. Vo of the column was 61 ml. Column dimensions were 2.5 x 37 cm. Ve/Vo values were 1.0, 1.6, 2.3 and 2.8 for peaks 1,2,3 and 4, respectively. 95 difference between Weber-Edsall extracts of washed muscle residue and those of prepared myofibrils. Peak 1 in Figure 35 has a Ve/Vo value of 1.0. This peak occasionally appeared heterogeneous as illustrated by the shoulder shown in Figure 35. Assuming that separation is occurring solely on the basis of gel filtration, a component with ve/Vo = 1.0 should have a molecular weight of $0,000,000 or larger (Bio-Rad Laboratories, 1968). Peak 2 of Figure 35, having a ve/Vo value of 1.6, (is relatively obscure, although it was frequently more pronounced. Molecular weight- elution volume data indicated that this peak had a molecular weight of approximately 5,000,000 (Bio-Rad Laboratories, 1968). The V e/Vo value of component 3 (Figure 35) is 2.3, indicating a molecular weight of around 200,000. The Ve/Vo ratio of 2.8 for fraction IV (Figure 35) lies outside the range of those values listed as obtainable by gel filtration (Bio-Rad Laboratories, 1968) Thus, component IV may have been retarded by absorption effects and its molecular weight cannot be estimated by gel filtration. Typical density gradient separation patterns from Weber-Edsall extracts of washed muscle residue and of prepared myofibrils are shown in Figures 36 A and B, respectively. Unlike gel filtration, density gradient centrifugation revealed large and consistent differences between the extracts of washed muscle residue and those of prepared myofibrils. Components 1 and 2 (Figures 36 A and B), with sedimentation distances of 1.1 cm and 2.3 cm, respectively, were present in preparations of both myofibrils and the washed nuscle residue. Components 3, 4 and 5 with sedimentation distances of 4.0, 5.8, and 6.5, respectively, were present in the washed muscle residue, but were largely absent from preparations . 0.10. 3 3H 1 2 - H v 4 Ln . N + r— 0.05.. 4 < . 5 2 . 2 3 o H q LLI 0.00 t I t I . I ' I T j o 2 4 6 8 10 :; "I 1 E d <- I.“ N a l 2 '— 0005‘ . .1 i h -. f4 ‘ i- " 'rl LiJ g'_.ad Figure 40. Disc gel electrophoresis of Weber-Edsall extract. Sample in each case was 0.05 ml of extract containing 1.5 mg protein/ml. A, stained with Amido Black. B, stained with DDD. Figure 41. Disc gel electrophoretic comparison of Weber-Edsall extract and actomyosin. A, sample = 0.05 ml of Weber-Edsall extract containing 1.5 mg protein/ml. B, sample = 0.05 ml of actomyosin preparation containing 1.8 mg protein/ml. 104 in the presence of urea suggests that urea may not actually inhibit actomyosin formation. It is possible that the dissociation prOperties of actin and myosin may be different for purified proteins and in myofibrils. Further investigations will be necessary to establish the dissociation properties under such conditions. Gel filtration of actomyosin produced the chromatogram shown in Figure 42. Assuming that separation occurred.solely on the baSis of gel filtration, peak 1 of Figure 42 consisted of material with a.molecular weight in the range of $0,000,000 or larger; whereas peak 2 had a molecular weight of about 6,000,000 (Bio-Rad Laboratories, 1968). Obviously, the two fractions separated by gel filtration are aggregates of several proteins and would not be expected to have molecular weights in agreement with values fer the various purified components. The nature of peaks 1 and 2 was further investigated by disc gel electrophoresis. Serial disc gel separations performed on the gel fil- tration fractions (Figure 42) are presented in Figure 43. The character- istic darkening of the myosin bands by the gel filtration process has been observed and discussed.previously herein. Peak 1, eluted at an effluent volume of 50 to 70 ml, appeared to consist largely of'nonrmigrating material (Rm = 0.00), myosin (Rm = 0.05-0.15) and actin (Rm = 0.39). However, peak 2, which was eluted at effluent volumes of 80, 90 and 100 ml, contained no aggregated material at Rm = 0.00. Thus, peak 2 contained.myosin (Rm = 0.05-0.15), actin (Rm = 0.39), reduced tropomyosin (Rm = 0.51) and an unidentified component at Rm = 0.59. The latter component appears to be identical to the band at Rm - 0.59 t 0.01, which has been observed earlier herein to occur in preparations of HM, PM, AA and IF (Table I). Attempts to identify this band were unsuccessful in the present study. 105 :5 0.10 l 2 <3 J l-fi . N F— 0.05 — ‘E E ‘ 1 C) . 2 -v J Lu 0.00 l T r I 5 I T I . 1 0 50 100 150 200 250 EFFLUENT VOLUME - ML. Figure 42. Gel filtration of actomyosin. A 2 ml sample of actomyosin preparation containing 2.2 mg protein/ml was applied at 0 ml effluent volume. Column dimensions were 2.5 x 38 cm. V0 of the column was 63 m1. Ve/Vo ratios for peaks 1 and 2 were 1.0 and 1.5, respectively. Rm ~ 3;ng a- .4- .s.' .‘_ l" u .2 J .9 J .5 J .l .1 R“ Figure 43. Serial disc gel electrophoretic analyses of the gel filtra- tion pattern from actomyosin shown in Figure 42. At far left, a 0.05 ml actomyosin sample containing 2.2 mg protein/ml was analyzed. Then, proceeding from left to right, 0.10 ml samples taken at 50, 60, 70, 80, 90 and 100 m1 effluent volume were analyzed. 106 The presence of reduced tropomyosin in peak 2 of Figure 42 provided direct evidence that tropomyosin is an integral part of the large aggre- gates contained in this fraction.7 Tropomyosin, applied.by itself to the column, was fOund to have a ve/Vo ratio of 2.2 fOr the oXidized species and 2.7 fer the reduced species; whereas, the Ve/Vo ratio of peak 2 is 1.5. Thus, tropomyosin appears to be an integral part of the purified actomyosin complex, apparently as a large aggregate. This was shown to be true since neither of the gel filtration peaks corresponded to the ‘Ve/Vb ratio of tropomyosin, yet tropomyosin was present in the disc gel pattern of peak 2. Similar determinations could not be reliably made for actin and myosin, apparently due to spontaneous aggregation of these purified.proteins on the gel column. Sucrose density gradient centrifugation of actomyosin usually produced two peaks as shown in Figure 44A. Peaks 1 and 2 sedimented at 1.1 and 2.0 cm, respectively. Johnson and Rowe (1964) have speculated on the nature of the two peaks produced by actomyosin in the ultracentringe. They suggested that the two peaks may result from two different types of actin participating in the actomyosin complex. In the present study, the nature of peaks 1 and 2 from ultra- centrifugation of purified actomyosin was investigated.by disc gel elec- trophoresis. Serial disc gel analyses of the separation achieved by density gradient centrifugation are shown in Figure 45A. The ultra- centrifuge pattern from 0.0 to 1.0 cm consisted mostly of oxidized tropo- myosin (Rm = 0.34 t 0.02). The sharp rise in 5245 at 1.1 cm (peak 1, Figure 44A) was accompanied by the appearance in the disc gel pattern of myosin (Rm = 0.05-0.15), actin (Rm = 0.39), reduced tropomyosin (R,m = 0.51), an unidentified band at Rm_= 0.60 t and.extra.protein fraction IA. Thus, 107 :8 H euefisucou ucoqmouw ecu oHQEmm .o 28 H eucamuaou ucuaeouu one season .m was madame sow: codumumoom douucoo .< cfim0%EOuum HE 0.0 m .ummu sumo GH .3; :3 5.8 .mfiowz 25 A new Ao.n may oumnomonoouan anamom .ouoo n35; o o con 38332; a: n mm me woaxo.m o om oono.u o w nona.n .moaxo.m o con unauaao pond: mam e as me moauo.o o om woaxn.a o w 1:qu “Sue; a can 3316 use: a: n me an ocflxo.a 0 cm «caxo.n c Q unanw>ouoo soda; 0 0 can 308333 a: m sen Non qo~uo.~ no~xo.~ om noaxo.m a w «Haaoouu gonad o :3 o can «flag a: H Amado omv Amman omv vuuuasoooH,Houuuoo oawH dads auuuuwuo uuaaa< .oz cusouu wouw< unaoo ulnuouuun ocuuuaaonH ounu0um coauoaauoom Huwuououm nouw< wuucuuaum #uauaom snow «0 uaouuom ouuoaauomuu HuouuoHo«aouo«z .HH canon 120 .a and h .oz ouaoaunonum magnum o>auouomouq no: owsmauuuoouuqurt cannon Houuaoo huuuo nu vo>hoano unwuoa owwuu>o a vouufisooau com a« vo>uooao n «o o u o I canon» Huguououn Hound uuuaauauu uncouom agenda Houuaou media a“ vo>hoano unwwon owuuobw 0H m Houuuoo «clam u« on once ad Ho: 0 I o>u I moans hound unaunqaou uaoouon ”unwanaouuusou ecu Beau cocuahuuov one: unwounuuuom .H announu dwe auuauuh a“ so m.o can w.n .o.¢ an wuauuuaqvoc «Moon no ugwuon ouuuo>w Huauwau no uoouuumt at at acnxw.¢ aged om oonN.n o a acmwowwoamau «enum.n m o ODOH Houownoaoucud «a so vane vane om neaxo.m o m usouas ..Sxm. a n o 003 2.8383: m: m r. I woos. n o 8. I! ‘ hoax¢.n o w waoauwuoa «a nonn.H o c coca wouounoaounu< «a «a cane o om «o~x~.~ o w usouau eonn.n o o coo” unaccoouoa: agenda 5 Amman omv Ammae cmv vouuasuooH Houuooo uaua mans Hawaamuo Huaaod .oz sunbuu wcww< ucaoo Huauouuum acupunoooH unencum cauuwaaoooH Huauouuon Houw< maauuuaum «ucwwum xuom we ucoouum Au.:oov HH «Hana 121 ° 0.10 :3 1 2 ‘3 m 4 cu . '— 0.05% < , E 1 Q 4 ._. 1 '-'-' 0.00 . 1 . r . ' , T r ‘ 0 7 4 6 8 10 SEDIMENTATION DISTANCE - CM. Figure 48A. Typical density gradient centrifugation pattern obtained from Weber-Edsall extract of fresh control muscle samples. Sample = 0.6 ml of Weber-Edsall extract. . 0.10 -« 2 ‘ E 4 g 4 N I I'—' 0.05 ‘n—1 < 4 E d o d J H L” 0.00 ' r ' l T I r I f 1 O 2 4 6 8 10 SEDIMENTATION DISTANCE - CM. Figure 483. Typical density gradient centrifugation pattern obtained from Weber-Edsall extract of control muscle samples after 20 days incubation at either 3 or 10°C. Sample = 0.6 m1 of Weber-Edsall extract. 122 ° 0.10 to D J 2 v d m 1 CU . I— 0.05 -' < . 2 Q 1 H LIJ 0.00 I f I ' I ' l ' I 0 2 4 6 8 10 SEDIMENTATION DISTANCE - CM. Figure 480. Typical density gradient centrifugation pattern obtained from Weber-Edsall extract of fresh inoculated muscle samples. Sample = 0.6 ml of Weber-Edsall extract. El CM AT 254 MU. 0 2 4 6 8 10 SEDIMENTATION DISTANCE - CM. Figure 480. Density gradient centrifugation pattern obtained from inoculated sample in Experiment No. l (inoculation performed with Streptococcus faecalis) after 20 days incubation at 3°C. Sample = 0.6 ml of Weber-Edsall extract. 123 E1 CM AT 254 MU. 0.00 v l m I . I v flit—1 0 2 4 6 8 10 SEDIMENTATION DISTANCE - CM. Figure 48E. Density gradient centrifugation pattern obtained from inoculated sample in Experiment No. 2 (inoculation performed with Pediococcus cerevisiae) after 20 days at 3°C. Sample - 0.6 m1 of Weber-Edsall extract. . 0.10- :3 2 v I.“ N ,_ 0.05- < 2 0 Fl Lu 0.00 0 2 4 6 8 10 SEDIMENTATION DISTANCE - CM. Figure 48F. Density gradient centrifugation pattern obtained from inoculated sample in Experiment No. 3 (inoculation performed with mixed culture from commercial meat) after 20 days of incubation at 3°C. Sample - 0.6 ml of Weber-Edsall extract. 124- El CM AT 254 MU. r T O 2 4 6 8 10 SEDIMENTATION DISTANCE - CM. Figure 480. Density gradient centrifugation pattern obtained from inoculated sample in Experiment No. 4 (inoculation performed with mixed culture from commercial meat) after 20 days of incubation at 3°C. Sample = 0.6 m1 of Weber-Edsall extract. 0.10- I:1 CM AT 254 MU. 0.00 v ‘ ' T '7 ‘7 ‘U '% 0 2 4 6 8 10 SEDIMENTATION DISTANCE - CM. Figure 4811. Density gradient centrifugation pattern obtained from inoculated sample in Experiment No. 5 (inoculation performed with Pediococcus cerevisiae) after 20 days at either 3 or 10°C. Sample - 0.6 ml of Weber-Edsall extract. 125 0.10- ‘J El CM AT 254 MU. 0.00 V r f I ' I ' I ' l 0 2 4 6 8 10 SEDIMENTATION DISTANCE - CM. Figure 481. Density gradient centrifugation pattern obtained from inoculated sample in Experiment No. 6 (inoculation performed with Pseudomonas fluorescens) after 20 days of incubation at either 3 or 10°C. Sample - 0.6 m1 of Weber-Edsall extract. 126 nature of these peaks was not ascertained in the present work.» The decrease in the height of these peaks would suggest that they were actually being used as nutrients by the bacteria. However, the possibility that the bacteria merely altered these fractions, thereby making them insoluble, was not ruled out in this study. In this regard, it is interesting to note the work ofLJay and.Kontou (1967) and Lerke §t_al, (1967), who presented evidence showing that the common spoilage bacteria of meat do not.use existing proteins for food, but instead.prefer small nonrprotein nitrogenous molecules. In the present work, disc gel electrophoresis was used to study the effects of bacterial growth on salt soluble proteins during Experiments No. S, 6, 7 and 8 (Table II). Typical disc gel patterns obtained during these experiments are shown in Figure 49. Results indicated no noticeable changes in the salt-soluble proteins during bacterial growth. The present microbiological experiments also penmit examination of species differences between salt-soluble proteins from the rabbit and the pig. Neither gel filtration nor density gradient centringation showed any consistent differences between the salt-soluble proteins of the rabbit and those of the pig. Disc gel electrophoresis (Figure 49) indicated that the salt-soluble proteins from pork had.the same or similar electrophoretic properties as those from.the rabbit. However, weber-Edsall extracts prepared from pig muscle consistently produced a darker band at Rm = 0.59 than those prepared from rabbit muscle. This band.appears to be identical with the unidentified component at Rm = 0.59, which has appeared in protein pre- parations throughout the course of the present study (Table I). The fact that Weber-Edsall extracts of pig muscle produce a darker band at Rm = 0.59 does not necessarily mean that this component is present in greater amounts in pig muscle. It could be more easily extracted from Figure 49. Disc gel electrophoresis of Weber-Edsall extracts obtained during microbiological experiments No. 7 and 8 (Table II). A, extracts were prepared on the day of inoculation. From left to right, extracts were obtained from Experiment No. 7 (rabbit) control, No. 7 (rabbit) inoculated with Achromobacter liquifaciens, No. 7 (rabbit) inoculated with Micrococcus luteus. No. 8 (pig) control, No. 8 (pig) inoculated with Achromobacter liquifaciens and No. 8 (pig) inoculated with Micrococcus luteus. B, extracts were prepared after 8 days incubation at 10°C. Distribution of samples is the same as in Figure 49A. C, extracts were prepared after 20 days incubation at 10°C. Distri- bution of samples is same as in Figure 49A. In each case. sample = 0.05 ml of Weber-Edsall extract, except in the case of the 8-day control from Experiment No. 7 (gel on far left in Figure 478), where 0.10 ml of extract was applied. 128 pig muscle, or it may have a greater affinity fer the Amido Black stain. Any one of these possibilities could.produce a darker band at Rm = 0.59. The feregoing experiments (Table II) indicated no detectable changes in the salt-soluble proteins of muscle during bacterial Spoilage. This is in agreement with the work of Jay (1966, 1967), Jay and Kontou (1967) and Lerke 33 a1, (1967), which indicated that the common meat spoilage organisms do not usually attack large protein.molecules, but instead utilize non-protein nitrogen sources. In the present work, both the aging process and bacterial growth were found to decrease the amount of certain non- protein, ultraviolet-absorbing components, which were detectable in the ultracentrifuge, but did not appear to be derived from the myofibrils. Results of disc gel electrophoresis indicated that the Weber-Edsall extract of pig muscle produced a more intense band at Rm.= 0.59 than that from rabbit muscle. SUMMARY The salt soluble proteins from skeletal muscle were separated and characterized by gel filtration, density gradient centrifugation, ion exchange chronatography and disc gel electrophoresis. Gel filtration and density gradient centrifugation afforded only crude fractionation, so that supplemental methods for separation were necessary. Ion exchange chromatography separated the Weber—Edsall extract of skeletal muscle into 8-11 fractions, but the method was too slow and cumbersome for routine analyses. Disc gel electrophoresis in the presence of 7 M urea was the most useful tool for fractionation and analyses of myofibrillar proteins, separating the Weber-Edsall extract into 8-11 principal bands. Each of the lmown myofibrillar proteins was isolated and purified by several methods. The purified proteins were then subjected to gel filtration, density gradient centrifugation and disc gel electrophoresis for further purification and characterization. Results showed that many of the so-called "pure preparations" of individual myofibrillar proteins contained significant ammmts of contaminants that could only be removed by special techniques. A comparison of four different procedures for preparing myosin indicated that chromatography on DEAE-Sephadex A- 50 gave the purest pre- paration. Disc gel electrophoresis in 8 M urea showed several bands at RIn values between 0.00 and 0.15. The material remaining at the origin probably consisted of myos in monomers and myosin aggregates. The bands 129 130 at Rma 0.05-0.15 were tentatively identified as being myosin poly- peptide sub-units. .Actin isolated directly from the myofibrils resulted in the purest actin preparation on comparing with three other isolation procedures. This preparation produced a single diffuse band at Rm = 0.39 by disc gel electrophoresis. Tropomyosin was prepared by feur different methods.- The purest preparation was obtained.by ammonium.sulfate fractionation and isoelectric precipitation followed by chromatography on DEAE-cellulose in the presence of 0.01 M EDTA. Two bands on disc gel patterns with Rm values of 0.34 and 0.50 were identified as tropomyosin. The slower moving component was apparently due to oxidation of the -SH groups, whereas, the faster moving component was reduced tropomyosin. Troponin (ESF) was prepared by two procedures. Disc gel bands were found in both preparations at Rm = 0.34 and 0.50, whidh corresponded to the oxidized and reduced forms of tropomyosin. This suggests that troponin was not separated from tropomyosin or else was nonrprotein in nature. Preparations of d-actinin, P-actinin, inhibitory factor and the extra protein group were made using only one method for each. Neither til-actinin, Q-actinin nor inhibitory factor were sufficiently pure to permit identification. . DEAE-cellulose chromatography of the extra.protein group separated it into four main fractions, which have been previously identified as Fractions I, II, III and IV on the basis of salt concentration required for elution.. A small peak following Fraction I was designated as Fraction IA. Fraction I was identified as consisting mainly of sarcoplasmic 131 proteins. Fractions II and III were. not identified, while Fraction IV contained considerable amounts of tropomyosin. Examination of the be- havior of Fraction IA indicates that it may be identical to troponin. Weber-Edsall extracts of washed muscle residue and of prepared myofibrils were compared. Although extracts of washed muscle residue contained non-proteinaceous canponents absorbing in the ultraviolet region, the amount and types of proteins appeared to be the same. Weber-Edsall extracts contained myosin, actin, oxidized and reduced tropomyosin, extra protein Fraction IA and probably °S-actinin. Specific staining of electro- phoretic gels of Weber-Edsall extracts indicated that the nucleic acids are complexed with tropomyosin, extra protein Fraction IA and possibly actin. In addition, Weber-Edsall extracts contained several unidentified components on disc gel electrophoresis, having relative mobilities of 0.15-0.30, 0.36 and 0.59. Actomyosin preparations contained myosin, actin, reduced trOpo- myosin, varying amounts of extra protein Fraction IA andprobably d-‘ac‘tinin. Several unidentified components were also present at relative mobilities of 0.36, 0.47 and 0.59 on disc gels. Actomyosin preparations contained two fractions detectable by gel filtration and density gradient centri- fugation. Gel filtration yielded two peaks with apparent molecular weights of $0,000,000 and 6,000 ,000. The former peak consisted mainly of actin and myosin, apparently aggregated together, while the second contained mainly actin, myosin, reduced tropomyosin and an unidentified component at RIn = 0.60. Density gradient centrifugation of actomyosin produced one fast- and one slow-sedimenting peak. The slow-sedimenting peak consisted of 132 myosin, oxidized tropomyosin, actin, reduced tropomyosin and an unknown component at Rm - 0.60. The fast-sedimenting peak consisted mainly of myosin, actin and reduced tropomyosin. Oxidized tropomyosin was not present in the initial actomyosin sample and appeared to have been formed during density gradient centrifugation. Results of gel filtration and density gradient centrifugation indicated that myosin, actin and tropo- myosin are all involved in the actomyosin complex. Pyrophosphate was fbund to have a general dissociating action on actomyosin, decreasing the sedimentation rate of all detectable protein moieties. Sedimentation behavior indicated that pyrophosphate caused an actual dissociation of myosin from the actomyosin complex, yet left actin and reduced tropomyosin in a partially dissociated or otherwise unnatural state. Pyrophosphate also decreased the sedimentation rate of an unknown component (disc gel electrophoretic Rm_= 0.50) and of extra.protein Fraction IA. Hewever, the extent of dissociation from.the actomyosin complex was not determined in the present study. The effect of EDTA on actomyosin was similar to the action of pyro- phosphate.) In general, EDTA appeared to dissociate myosin and partially dissociate actin and reduced tropomyosin. 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